Derivation of embryonic stem cells

ABSTRACT

This present invention provides novel methods for deriving embryonic stem cells, those cells and cell lines, and the use of the cells for therapeutic and research purposes without the destruction of the embryo. It also relates to novel methods of establishing and storing an autologous stem cell line prior to implantation of an embryo, e.g., in conjunction with reproductive therapies such as IVF.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of United States ProvisionalApplication Nos.: 60/624,827, filed Nov. 4, 2004; 60/662,489, filed onMar. 15, 2005; 60/687,158, filed Jun. 3, 2005; 60/723,066, filed on Oct.3, 2005 and 60/726,775, filed on Oct. 14, 2005. The disclosures of theseapplications are hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

This invention generally relates to novel methods for deriving embryonicstem cells, those cells and cell lines, and the use of the cells fortherapeutic and research purposes. It also relates to novel methods ofestablishing and storing an autologous stem cell line prior toimplantation of an embryo, e.g. in conjunction with assistedreproductive technologies such as in vitro fertilization.

BACKGROUND OF THE INVENTION

With few exceptions, embryonic stem cells have only been grown fromblastocyst-stage embryos. ES cell lines are conventionally isolated fromthe inner cell mass of blastocysts and in a few instances from cleavagestage embryos. There are several drawbacks to the techniques used tocreate these cells. From the perspective of the technique, the culturingof embryos to blastocysts occasionally has a relatively low successrate. Some people express the basic objection that embryonic stem (ES)cell research is rooted in the fact that ES-cell derivation deprivespreimplantation-stage embryos of any further potential to develop into acomplete human being. The following invention provides novel andunexpected methods of deriving embryonic stem cell lines and otherembryo-derived cells for use in research and in medicine.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows fibroblast-like cells originating from ES colonies that canbe expanded for use as autologous feeders.

FIG. 2 depicts blastomere-derived mES cells stained for Lac-Z using akit from Sigma (A). FIGS. 2B-2D show immunostaining for the same cellsexpressing both Oct-4 (2B) and Lac-Z(2C). FIG. 2D representscounterstaining with DAPI. Bar, 100 um.

FIG. 3 shows differentiation of blastomere-derived mES cells in vivo andin vitro. In FIG. 3A shows a mouse embryo that was fixed in 2%glutaraldehyde, 4% paraformaldehyde overnight and stained for Lac-Zusing a kit form Sigma. FIGS. 3B-3D show immunofluorescence staining formolecular markers of primitive endoderm (α-feto protein, 3B), ectoderm(βIII tubulin, 3C) and mesoderm (muscle actin, 3D). Bar, 100 um.

FIG. 4 illustrates stages of single blastomere growth in the presence(4A-4F) or absence (4G, 4H) of mES cells. FIGS. 4A (green fluorescence)and 4B (Hoffman modulation optics) visualize an aggregate of GFP mEScells 48 hours after aggregation with single blastomeres. The arrow inFIG. 4B shows a protruding cluster of GFP-negative cells not visible inFIG. 4A. FIGS. 4C (green fluorescence) and 4D (phase contrast)demonstrate outgrowth of GFP-negative cells aggregated with GFP⁺ mEScells, after being plated on mouse embryo fibroblast cells (MEF). Thearrows in FIGS. 4C and 4D point to GFP-negative cells. FIGS. 4E (greenfluorescence) and 4F (phase contrast) display growth of GFP⁺ mES cellsand cells arising from a single blastomere after mechanical dissociationof initial outgrowth. The arrows in FIGS. 4E and 4F point to remainingGFP⁺ mES cells. FIG. 4G represents cells derived from a singleblastomere grown on MEF alone for four days without ES cells, stainedwith Tromal, which labels trophoblast cells. FIG. 4H shows the samecells as Figure G, but are stained with DAPI to show the three nuclei.Scale bar, 100 um.

FIG. 5 shows PCR analysis of LacZ, GFP, and stem cell marker genes inembryonic stem and trophoblast stem (“TS”) cell lines. FIG. 5A providesPCR analysis using LacZ-specific primers demonstrating the presence ofthe LacZ gene in the ES and TS cell lines. FIG. 5B shows PCR analysisfor GFP-specific primers showing the absence of helper-ES cell (GFPpositive) contamination. In FIG. 5C, RT-PCR analysis reveals robustexpression of the Oct-4 gene (5C) in the ES cell lines, but much lowerlevels in the TS cell lines. The TS cell lines showed a large PCRproduct in addition to the expected fragment. Figure 5D representsanalysis of nanog gene demonstrating moderate to high levels ofexpression in the ES cell lines, and moderate levels in the TS celllines. FIG. 5E shows similar levels of Rex-1 gene expression in ES andTS cells lines. FIG. 5F shows high levels of trophoblast marker Cdx-2gene expression in the TS cell lines, and low to negligible levels inthe ES cell lines. FIG. 5G shows α-Tubulin used as a control for theinput of RNA samples. The abbreviations present in FIG. 5 are asfollows: M, molecular weight marker; LacZ, genomic DNA isolated from129/Sv-ROSA26:LacZ mouse tails; GFP, genomic DNA isolated from greenfluorescent protein (GFP)-positive 129Sv/CD-1 mouse ES cells; CD-1,genomic DNA isolated from CD-1 mouse tails; H, H₂O control. PL, mouseplacental RNA, M, molecular weight marker; H, H₂O control.

FIG. 6 illustrates a comparison of putative ES (left column) and TS(right column) cell lines derived from single blastomeres. FIGS. 6A and6B show phase contrast photos of typical colonies. FIGS. 6C and 6Drepresent Lac-Z stained colonies, demonstrating their single blastomereorigin. FIGS. 6E and 6F show alkaline phosphatase staining. FIGS. 6G and6H show indirect immunofluorescence with antibodies to Oct-4. FIG. 6Idepicts putative ES cells stained with antibodies to SSEA-1. FIG. 6Jshows TROMA-1 antibody staining of the putative TS cells (same field asFIG. 6H). Scale bar, 200 um.

FIG. 7 shows LacZ stained placenta of 10.5 day chimera showingcontribution of single blastomere-derived TS cells. The maternal portionof the placenta has been peeled away. In this figure, the embryonicportion shown is photographed from the distal side of the disk and isapproximately 4 mm in diameter.

FIG. 8 depicts differentiation of blastomere-derived mES cells in vitroand in vivo. FIGS. 8A-8C show immunofluorescence analysis of molecularmarkers of mesoderm (muscle actin, FIG. 8A), primitive endoderm (α-fetoprotein, FIG. 8B), and ectoderm (β III tubulin, FIG. 8C). FIG. 8Ddepicts representative chromosome spreads of two singleblastomere-derived mES cell line. G-banded karyotyping shows that linesY1 (top) and Y7 (bottom) have XY and XX karyotypes, respectively. FIG.8E shows hematoxylin and eosin stained section through a teratoma andshows examples of tissue from all three germ layers. Bn, bone(mesoderm); nt, neural tissue (ectoderm); cre, ciliated respiratoryepithelium (endoderm). The insert of FIG. 8E is an enlarged region ofciliated respiratory epithelium. FIG. 8F shows 11.5 day chimeric embryosproduced from three of the putative ES cell lines, each of which showsthe high degree of chimerism frequently observed. FIG. 8G is a closeupview of lacZ-stained chimera from putative ES line J-15. The arrowpoints to the placental labyrinth, which is also chimeric and is derivedfrom extraembryonic mesoderm, not trophectoderm. FIG. 8H displayschimeric pups generated by aggregating blastomere-derived (1 29/Sv) EScells (lines J15 and Y1) with CD-1 mouse embryos. Scale bars: A-D—200um, F—10 mm, G—2 mm.

FIG. 9 shows PCR analysis demonstrating the presence of the LacZ gene inpurified sperm from chimeric mice produced from two differentblastomere-derived ES cell lines. The abbreviations present in FIG. 9are to be understood as follows: M, molecular weight marker; H, H₂Ocontrol; ES, DNA from mouse ES cells used to generate chimeric animals;CD-1, DNA from CD-1 mouse; SP-1, DNA from sperm of chimeric mouse No. 1;SP-2, DNA from sperm of chimeric mouse No. 2.

SUMMARY OF THE INVENTION

The present invention provides novel methods for deriving embryonic stemcells, those cells and cell lines, and uses of the embryonic stem cellsand cell lines for therapeutic and research purposes. It also relates toa method of establishing and storing an autologous stem cell line from ablastomere retrieved prior to implantation of an embryo, e.g. inconjunction with assisted reproductive technologies such as in vitrofertilization (“IVF”).

This invention provides a method of producing an embryonic stem cell,comprising the step of culturing a blastomere obtained from an embryo,wherein the embryo remains viable. In one embodiment, the blastomere isobtained from an embryo prior to compaction of the morula. In anotherembodiment, the embryo is obtained before formation of the blastocoel.The blastomere may be obtained by partial or complete removal of thezona pellucida surrounding the embryo. The embryo may be implanted orcryopreserved.

The blastomere obtained from the embryo is cultured with any suitablecell to produce an ES cell. Cells suitable for culturing the blastomeresinclude, but are not limited to, embryonic stem cells, such as fromalready established lines, embryo carcinoma cells, murine embryonicfibroblasts, other embryo-like cells, cells of embryonic origin or cellsderived from embryos, many of which are known in the art and availablefrom the American Type Culture Collection, Manassas, VA 20110-2209, USA,and other sources. The blastomere may also be cultured with factors thatinhibit differentiation of the ES cell. In one embodiment, theblastomere is cultured in the presence of heparin. In anotherembodiment, Oct-4 is introduced into the blastomere or alternatively,expression of endogenous Oct-4 is induced in the blastomere.

In one embodiment, the present invention provides a method of producingan ES cell comprising the steps of obtaining a blastomere from anembryo, wherein the embryo remains viable, aggregating the blastomerewith ES cells, culturing the aggregated blastomere and ES cells untilthe blastomere exhibits properties of ES cells, and isolating the EScells derived from the blastomere.

In another embodiment, the blastomere obtained from an embryo iscultured with autologous feeder cells, wherein the feeder cells areproduced by culturing a blastomere obtained from the same embryo underconditions to differentiate the blastomere into a somatic cell toproduce the autologous feeder cells.

In a further embodiment, a blastomere obtained from an embryo undergoescell division and one progeny cell is used for genetic testing andanother progeny cell is used to produce an ES cell.

In one embodiment, the method of producing an ES cell or ES cell linecomprises obtaining a blastomere through biopsy, removing the zonapellucida, separating the blastocyst into two segments, culturing oneblastocyst segment in order to produce an ES cell or ES cell line andimplanting or cryopreserving the remainder of the blastocyst. In anotherembodiment the method comprises the steps of obtaining a singleblastomere prior to implantation and before formation of the blastocoel,culturing the blastomere, adding ES cells from already establishedlines, allowing the ES cells to clump around the blastomere until theblastomere exhibits ES cell growth and harvesting the resultant ES cellfor therapeutic purposes. In yet another embodiment, the methodcomprises the steps of obtaining a single blastomere before compactionof the morula, culturing the blastomere in standard culture conditions,adding mitotically inactivated ES cells from already established linesuntil ES cells begin to form, and harvesting or cryopreserving theresultant ES cells. In a further embodiment, the method comprises thesteps of obtaining a single blastomere prior to implantation and beforeformation of the blastocoel, culturing the blastomere, adding ES cellsfrom already established lines, and introducing recombinant Oct-4 intothe blastomere or activating endogenous Oct-4.

The ES cell produced from the blastomere may be pluripotent ortotipotent. Pluripotency or totipotency of the ES cell may be determinedby assaying for ES cell marker proteins. Such proteins include Oct-4,SSEA-1, nanog, alkaline phosphatase and Res-1.

The method of the invention may be performed on mammals, e.g., mice,rabbits, sheep, pigs, cows, primates and humans. In one embodiment, themammal is a non-human mammal. In another embodiment, the mammal is ahuman.

The present invention also provides methods of differentiating the EScells produced by the methods of the invention. The ES cells may bedifferentiated into any cell type including those of mesodermal,endodermal and ectodermal origin.

Also contemplated are methods of differentiating the blastomere obtainedfrom an embryo into a differentiated cell type, e.g., mesoderm, endodermor ectoderm without first producing an ES cell from the blastomere.

The invention also encompasses the ES cells produced by the methods ofthis invention, ES cell lines derived from these ES cells as well asdifferentiated cells derived from the ES cells or cell lines.

The ES cells provided by this invention or cells derived from the EScells are useful for treating disorders amenable to cell therapy.Pharmaceutical compositions comprising these cells together with apharmaceutically acceptable medium or carrier are also provided.

Also provided are methods of producing trophoblast stem (TS) cellscomprising the step of culturing a blastomere obtained from an embryo,wherein the embryo remains viable. In one embodiment, blastomere isobtained prior to compaction of the morula. In another embodiment, theblastomere is obtained before formation of the blastocoel. Theblastomere may be obtained by partial or complete removal of the zonapellucida surrounding the embryo.

The blastomere obtained from the embryo is cultured with any suitablecell to produce a TS cell. Cells suitable for culturing the blastomeresinclude, but are not limited to, embryonic stem cells, such as fromalready established lines, embryo carcinoma cells, murine embryonicfibroblasts, other embryo-like cells, cells of embryonic origin or cellsderived from embryos, many of which are known in the art and availablefrom the American Type Culture Collection, Manassas, VA 20110-2209, USA,and other sources. The blastomere may also be cultured with factors thatinduce differentiation of the ES cell. In one embodiment, the blastomereis cultured in the presence of FGF-4.

The TS cell produced by the methods of the invention may express a TScell marker, e.g., nanog, Rex-1, cdx-2. The TS cell may also lackexpression of Oct-4 or α-fetoprotein. The TS cell may also be culturedto produce a TS cell line or differentiated.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based in part on the discovery that stem cellscan be generated from embryos without affecting viability of the embryo.In one embodiment, these methods utilize in vitro techniques currentlyused in pre-implantation genetic diagnosis (PGD). As demonstratedherein, pluripotent embryonic stem (ES) cell lines can be generated froma single blastomere removed from an embryo without interfering with theembryo's normal development to birth.

Removal of the Blastomere

The blastomere may be removed from an embryo at various developmentalstages prior to implantation including but not limited to: beforecompaction of the morula, during compaction of the morula, beforeformation of the blastocoel or during the blastocyst stage.

In one embodiment the invention provides methods for biopsy of ablastocyst which will produce embryonic stem cells, and the remainder ofthe blastocyst is implanted and results in a pregnancy and later in alive birth. In an example of this: the zona pellucida is removed fromthe blastocyst by any means known to those of ordinary skill in the art(in this instance using acidic tyrode solution pH 2.4), it is placed onculture ware with protein free media (CZB protein free media pH 7.4 isused, but other protein free media could be used) and it adheres, thenthe blastocyst is biopsied. This was done using a small segment of razorblade attached to a pipette, and it was cut once separating theblastocyst into two segments—preferably less than 30%, 20%, 10% or 5% ofthe blastocyst is biopsied. Serum is then added to the media todissociate the blastocyst and the biopsied segment is used to deriveembryonic stem cells or other embryo-derived cells through means wellknown to those of ordinary skill in the art (e.g., allowing the biopsyto grow on embryonic fibroblasts, on feeder-free matrix, etc.) while theremainder of the blastocyst is implanted or cryopreserved.

In another embodiment the controversies associated with the derivationof embryonic stem cells are circumvented by using a technique similar tothat used in preimplantation genetic diagnosis (PGD) where a singleblastomere is removed from the embryo, preferably before the compactionof the morula. These methods can be adapted for use in the presentinvention, for the removal of one or more cells from an embryo withoutaffecting the continued development of the embryo. In one embodiment,the biopsied blastomere is allowed to undergo cell division and oneprogeny cell is used for genetic testing and the remaining cells areused to generate stem cells. The biopsied embryo may be implanted at theblastocyst stage or frozen for implantation at a later time.

The biopsy consists of two stages. The first is to make a hole in, or insome instances fully remove, the zone pellucida that surrounds theembryo. Once the hole is made, the cells (preferably one or two) maythen be removed from the embryo. In certain preferred embodiments, themethod involves removing or generating an extraction hole in the zonapellucida, and can be carried out by one more techniques selected fromthe group consisting of physical manipulation, chemical treatment andenzymatic digestion. Exemplary techniques that can be used include:

-   -   Partial zone dissection (PZD:): partial dissection of the zona        pellucida, using a micro-pipette;    -   Zona drilling: chemical opening of the zona pellucida zone        through partial digestion with Tyrode acid;    -   Zona drilling: enzymatic opening of the zona pellucida zone        through partial digestion with pronase or other protease;    -   zona pellucida thinning: thinning of the zona pellucida with        Tyrode acid or laser;    -   Point-like opening of the zona pellucida with laser;    -   Point-like mechanical opening of the zona pellucida with Piezo        micro-manipulator.

To briefly illustrate one embodiment, the procedure is performed on day3 of embryo development, when the embryo is around 6-8 cell stage. Theembryo is placed in a drop of biopsy medium under mineral oil by holdingit with a holding pipette. The zona pellucida is locally digested, byreleasing acidified Tyrode's solution (Sigma, St. Louis, Mo. 63178)through an assistant hatching pipette. Once the hole is made, cells(blastomeres) can be aspirated through the hole.

To illustrate another embodiment, the zona pellucida of the blastocystmay be at least partially digested by treatment with one or more enzymesor mixture of enzymes such as pronase. A brief pronase (Sigma) treatmentof blastocysts with an intact zona pellucida results in the removal ofthe zona. Other types of proteases with the same or similar proteaseactivity as pronase may also be used.

Culturing the Blastomere

The isolated blastomeres may be cultured by placing them on cultureware(e.g., in microwells) with media in standard culture conditions togetherwith any suitable cells including but not limited to embryonic stemcells, such as from already established lines, embryo carcinoma cells,murine embryonic fibroblasts, other embryo-like cells, cells ofembryonic origin or cells derived from embryos, many of which are knownin the art and available from the American Type Culture Collection,Manassas, VA 20110-2209, USA, and other sources. These cells clump oraggregate around the blastomere. Other methods of aggregation includingmethods using microwell microbeads or the hanging drop method, or anyother aggregation method known in the art may be used. While not wishingto be bound by any particular theory, it is believed that over a periodof days or weeks the cultured blastomeres exhibit ES cell growth perhapsas a result of cell-cell interactions between the blastomeres and theco-cultured embryonic cells or from interactions between the blastomeresand factors secreted by the embryonic cells.

The blastomere(s) may be co-cultured with the remaining embryo. In oneembodiment, the blastomeres are co-cultured with the remaining embryo ina microdroplet culture system or other culture system known in the art,which permits cell-cell, cell-secreted factor and/or cell-matrixcontact. The volume of the microdrop may be reduced, e.g., from 50microliters to about 5 microliters to intensify the signal and promotecell-cell interactions.

In certain embodiments, the blastomere culture conditions may includecontacting the cells with factors that can inhibit or otherwisepotentiate the differentiation of the cells, e.g., prevent thedifferentiation of the cells into non-ES cells, trophectoderm or othercell types. Such conditions can include contacting the cultured cellswith heparin or introducing Oct-4 into the cells (such as by includingOct-4 in the media) or activating endogenous Oct-4 in the cells.

Autologous Feeder Cells

The present invention also provides a method of plating earlypre-blastocyst embryos to make stem cells on autologous feeder cells. Inone embodiment, this method comprises (a) splitting a pre-blastocystembryo, (b) plating one part into tissue culture under conditions todirectly differentiate it into somatic cells to make feeder cells and(c) plating the other part of the pre-blastocyst embryo on theautologous feeder cells. In another embodiment, the autologous feedercells and ES cells are produced from blastomeres removed from thepre-blastocyst embryo, thus, preserving the ability of the embryo to beimplanted.

Pluripotency of ES Cells

Pluripotency of the ES cells produced by the methods of this inventioncan be determined by detecting expression of ES cell marker proteins.Examples of such proteins include but are not limited to octamer bindingprotein 4 (Oct-4), stage-specific embryonic antigen (SSEA)-1, nanog,alkaline phosphatase and Res-1. In some embodiments, the putative EScell lines maintain pluripotency after more than 13, 20, 30, 40, 50, 60,70, 80, 90 or 100 passages. The ES cells may also be assayed formaintenance of normal karyotype.

Production of TS Cells

This invention also provides methods of producing trophoblast stem (“TS”cells) by contacting blastomere outgrowths, which morphologicallyresemble trophoblast and/or extraembryonic endoderm, but which do notresemble ES cells, with FGF-4. For example, FGF-4 is added to theculture media of the outgrowths. TS cells can be detected by assayingexpression of proteins such as nanog, Rex-1, and Cdx-2, using proceduresstandard in the art. TS cell identification can also be evidenced byabsence of the expression of proteins such as, but not limited to Oct-4and α-feto protein.

Therapeutic Uses of ES Cells

The present invention provides a method of treating a disorder amendableto cell therapy comprising administering to the affected subject atherapeutically effective amount of the ES cells of the invention. TheES cells of this invention are suitable for any use that ES cells areuseful.

In one embodiment the methods of the invention are used to remove ablastomere preceding implantation of an embryo after which theblastomere would be cultured as described above in order to derive andstore embryonic stem cells for therapeutic uses using cell therapyshould the child resulting from the embryo require, for example, diseasetherapy, tissue repair, transplantation, treatment of a cellulardebilitation, or treatment of cellular dysfunctions in the future.

In another embodiment of the invention, cells derived from a blastomere,precompaction morula, compacting morula, or sectioned blastocyst aredirectly differentiated in vitro or in vivo to generate differentiatingor differentiated cells without generating an embryonic stem cell line.These embryo-derived cells, like embryonic stem cells are useful inmedical and biological research and in the treatment of disease byproviding cells for use in cell therapy, e.g., allogeneic cell therapy.

The embryonic stem cells and embryo-derived cells generated by theabove-mentioned novel techniques are utilized in research relating tocell biology, drug discovery, and in cell therapy, including but notlimited to production of hematopoietic and hemangioblastic cells for thetreatment of blood disorders, vascular disorders, heart disease, cancer,and wound healing, pancreatic beta cells useful in the treatment ofdiabetes, retinal cells such as neural cells and retinal pigmentepithelial cells useful in the treatment of retinal disease such asretinitis pigmentosa and macular degeneration, neurons useful intreating Parkinson's disease, Alzheimer's disease, chronic pain, stroke,psychiatric disorders, and spinal cord injury, heart muscle cells usefulin treating heart disease such as heart failure, skin cells useful intreating wounds for scarless wound repair, bums, promoting wound repair,and in treating skin aging, liver cells for the treatment of liverdisease such as cirrhotic liver disease, kidney cells for the treatmentof kidney disease such as renal failure, cartilage for the treatment ofarthritis, lung cells for the treatment of lung disease and bone cellsuseful in the treatment of bone disorders such as osteoporosis.

Such cell therapy methods may involve use of the ES cells of thisinvention in combination with proliferation factors, lineage-commitmentfactors, or gene or proteins of interest. Treatment methods may includeproviding stem or appropriate precursor cells directly fortransplantation where the tissue is regenerated in vivo or recreatingthe desired tissue in vitro and then providing the tissue to theaffected subject.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. In case of conflict, the present specification, includingdefinitions, will control. Further, unless otherwise required bycontext, singular terms shall include pluralities and plural terms shallinclude the singular. Generally, nomenclatures used in connection with,and techniques of, cell and tissue culture, molecular biology,immunology, microbiology, genetics, developmental biology, cell biologydescribed herein are those well-known and commonly used in the art.Exemplary methods and materials are described below, although methodsand materials similar or equivalent to those described herein can alsobe used in the practice or testing of the present invention.

All publications and other references mentioned herein are incorporatedby reference in their entirety. Although a number of documents are citedherein, this citation does not constitute an admission that any of thesedocuments forms part of the common general knowledge in the art.Throughout this specification and claims, the word “comprise,” orvariations such as “comprises” or “comprising” will be understood toimply the inclusion of a stated integer or group of integers but not theexclusion of any other integer or group of integers.

In order for that this invention may be better understood, the followingexamples are set forth. These examples are for purposes of illustrationonly and are not be construed as limiting the scope of the invention inany matter.

EXAMPLE 1 Generation of ES Cell Lines

Single blastomeres were isolated from 8-cell stage 129/Sv-ROSA26:LacZmouse embryos either by biopsy through a hole in the zona pellucidadrilled using Piezo-pulse or by disaggregating of zona-denuded embryosin Ca++/Mg++ free PBS for 10 minutes. The biopsied (7-cell) embryos weretransferred to the oviducts of 1.5 days post coitum (d.p.c.)synchronized surrogate mothers, and each separated blastomere aggregatedwith a small clump (approximately 100 cells) of green fluorescentprotein (GFP)-positive 129Sv/CD-1 mouse ES (mES) cells in a 300 umdepression created by pressing an aggregation needle into the bottom ofa plastic tissue culture plate. After incubation for 24-48h a growing“bud” of GFP-negative cells was observed on the sides of the majority(60%) of GFP-mES clusters (See FIG. 4 A, B). The cell clumps were platedonto mitomicin C-treated mouse embryonic fibroblasts (MEF) and culturedin knockout DMEM (15% FCS, penicillin/streptomycin, Glutamax-I,β-mercaptoethanol, nonessential amino acids, LIF [2000U/ml], and MEK1inhibitor [50 μM] (mES culture medium)). See, for example, Hogan et al.Manipulating the Mouse Embryos: A Laboratory Manual. Cold Spring HarborLaboratory Press; 2nd Edition, 1994. The majority of blastomeres (54/91)formed rapidly growing clumps of cells within 4 days, which wereseparated from GFP-positive mES cells under a fluorescence microscope.The cells were expanded by mechanical dissociation or trypsinization,while selecting for the colonies morphologically resembling ES cells andexcluding any GFP-positive cells (FIG. 4, C, D, E, F).

Several lines of LacZ positive ES-like cells were produced (Table 1,FIG. 6 C) which maintained normal karyotype (FIG. 8 D) and markers ofpluripotency after over 50 passages. Each line expressed octamer bindingprotein 4 (Oct-4), stage-specific embryonic antigen (SSEA)-1, nanog, andalkaline phosphatase (FIG. 2 and FIG. 6 E,G,I). Indirectimmunofluorescence staining for ES cell protein markers was performed oncells growing on 4-well tissue culture plates. For example, see Lanza R,et al, Eds. Handbook of stem Cells. Vol 1:Embryonic Stem Cells(Elsevier/Academic Press, San Diego, Calif., 2004); Evans, M. J.,.Kaufman, M. H., Nature 292, 154 (1981); Thomson JA et al., 282, 1145(1998); Cowan C. A. et al., N. Engl J Med. 350, 1353 (2004). Thefollowing primary antibodies were used: Oct-4 (Santa Cruz Biotechnology,Santa Cruz, Calif.), SSEA-1 (developed by Solter and Knowles andobtained through the DSHB of the University of Iowa, Iowa City, Iowa),Troma-1 (raised by Brulet and Kemler and obtained through DSHB), α-fetoprotein (DACO), βIII tubulin (Covance, Berkeley, Calif.) and muscleactin (Abcam, Cambridge, Mass.). Alkaline phosphatase staining wasperformed using the Vector Red kit from Vector Laboratories.

Polymerase chain reaction (PCR) analysis revealed the presence of LacZbut not GFP gene sequences in these cells (FIG. 5 A, B), confirming thatthe lines originated from the blastomeres and not the ES cells used foraggregation. Briefly, genomic DNA was isolated from ES and TS cellsusing a QIAamp DNA Mini Kit (Qiagen, Valencia, Calif.), and 100 ng perreaction was used for both GFP and LacZ gene amplification. We usedforward (5′-TTGAATTCGCCACCATGGTGAGC-3′) (SEQ ID NO:1) and reverse(5′-TTGAATTCTTACTTGTACAGCTCGTCC-340 ) (SEQ ID NO:2) primers for GFP genewith reaction parameters of 95° C. for 9 min (1 cycle) and 94° C. for 45s, 59° C. for 1 min, 72° C. for 1.5 min for 37 cycles. PCR products wereseparated on 1.5% agarose gel and visualized by ethidium bromidestaining. LacZ gene genotype analysis was performed with primers and PCRparameters recommended by The Jackson Laboratory (Bar Harbor, Me.).

In two control experiments, individual blastomeres (n=44) isolated from8-cell embryos were plated into 20-100 μl drops containing mES cellculture medium. The majority of the blastomeres failed to divide overthe 10 day period of culture, whereas 9 (20%) generated small clustersof differentiated trophoblast-like giant cells (FIG. 4 G, H) beforearresting at the 2- to-6 cell stage. This suggests that cell co-cultureor the exposure of the blastomeres to substances secreted by the EScells may be critical to the success of this method.

EXAMPLE 2 Differentiation of ES Cells

When the ES-like cell cultures were allowed to overgrow, theyspontaneously differentiated into cells of all three germ layers, asevidenced by immunostaining with antibodies to muscle actin (mesoderm),βIII tubulin (ectoderm), and α-feto protein (primitive endoderm)(FIG. 3B-D and FIG. 8 A-C). Beating heart muscle, extraembryonic endoderm andmultiple neuronal cell types were also routinely observed indifferentiating cultures. To further demonstrate the pluripotency of thederived ES cells, ES cell lines were either injected into CD-1 mouseblastocysts or aggregated with 8-cell stage morulae as describedpreviously (Hogan et al., supra) and transferred to recipient females.X-Gal (5-bromo—4-chloro—3-indolyl-beta-D-galactopyranoside) staining ofthe resulting chimeric fetuses showed that the ES cell lines contributedto all organs (FIG. 3 A), such as, heart, kidney, liver, lung,intestine, brain, blood, skin and genital ridge. Twenty-four of thefetuses (83%) were chimeric (FIG. 8 F, G), and eight of nine (89%) pups(FIG. 8H) were chimeric; (the latter had the LacZ gene in their gametes(confirmed by PCR analysis; FIG. 9), and produced LacZ+ offspring whencrossed with CD-1, confirming the contribution of the blastomere-derivedES cells to the germ line.).

To further analyze the pluripotency of the ES cells, the ES cells wereinjected into NOD-SCID mice and examined for their ability todifferentiate into various cell types. Briefly, approximately 1 millionES cells were injected into the rear thigh of a NOD-SCID mouse. Afterabout two months the mice were sacrificed and the teratomas excised,fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. Theteratomas contained tissues from all three germ layers including boneand cartilage (mesoderm), neural rosettes (ectoderm), and ciliatedrespiratory epithelia (endoderm) among others (FIG. 8E).

No. non- GFP outgrowths No. non- Differentiation detected GFP ES linesNo Exp. No. after plating outgrowths estab- Markers In vitro musclechimeras/ No. blastomeres on MEF passage 1 lished Oct-4 AP Nanog SSEA1Tb AFP actin fetuses Karyotype 1 22 14 6 0 2 24 13 8 0 3 24 13 6J15 + + + + + + + 7/9 40XY 4 21 16 5 Y1 + + + + + + + 5/5 40XY 5 15  8 5Y7 + + + + + + + 8/9 40XX 6 19 11 6 J5 + + + + + + + 4/6 60XXY 6 — — —Y8 + + + + + + + — 40XX

The blastomere-biopsied embryos developed to term without a reduction intheir developmental capacity (49% [23/47] live young versus 51% [38/75]for control non-biopsied embryos (Chi-square test, p=0.85). Theseresults are consistent with human data, which indicates that normal andPGD-biopsied embryos develop into blastocysts with comparableefficiency. Although only 25 of 91 blastomeres (27%) generated innercell mass (ICM)-like outgrowth, and only a few stable ES stem-cell lineswere obtained in this study, we believe this success rate can beconsiderably increased by greater attention to the earliest stages ofblastomere outgrowth, as well as the use of various measures whichinhibit or influence the spontaneous differentiation of pluripotentcells into trophectoderm and other cell types.

These data show that ES cell lines can be derived without embryodestruction.

EXAMPLE 3 Generation of TS Cell Lines

Blastomere outgrowths that morphologically resembled trophoblast andextraembryonic endoderm but not ES cells were further cultured in themES cell medium with 50 ng/ml FGF-4 produced trophoblast stem (TS)-likecells that were maintained under these conditions and passaged withtrypsin. Seven putative TS lines were established, which maintainednormal karyotype and expressed markers of TS cells (FIG. 6 B, D, F, H,J). These cells were negative for Oct-4 (FIG. 6 H) and for α-fetoprotein. Putative TS cells contributed to the extraembryonic lineage inchimeric fetuses generated by aggregation with the LacZ⁺ TS-cells (FIG.7). RT-PCR analysis confirmed that these cells expressed Cdx-2, but notOct-4 (FIG. 5 C and F). Nanog and Rex-1 were expressed in both theputative TS and ES cell lines (FIG. 5 D and E).

Briefly, RT-PCR analysis was performed as follows: Total RNA wasisolated from ES and TS cells using an RNAeasy Mini Kit (Qiagen), and 1μg RNA was subjected to first strand cDNA synthesis with an oligo (dT)primer, using AMV reverse transcriptase (Promega, Madison, Wis.). Onetenth of the RT reaction was subjected to PCR amplification. PCRconditions for all genes were 95° C. for 9 min (1 cycle), 94° C. for 45s, 62° C. for 1 min and 72° C. for 1.5 min with 2 mM Mg++ concentration,except for α-tubulin gene that was annealed at 64° C. Primers used were:Oct-4 gene, forward 5′-CTGAGGGCCAGGCAGGAGCACGAG-3′ (SEQ ID NO:3),reverse 5′-CTGTAGGGAGGGCTTCGGGCACTT-3′ (484 bp) (SEQ ID NO:4); Nanoggene, forward 5′-GGGTCTGCTACTGAGATGCTCTG-3′ (SEQ ID NO:5), reverse5′-CAACCACTGGTTTTTCTGCCACCG-3 (363 bp) (SEQ ID NO:6); Cdx2 gene, forward5′-GGCGAAACCTGTGCGAGTGGATGCGGAA-3′ (SEQ ID NO:7), reverse5′-GATTGCTGTGCCGCCGCCGCTTCAGACC-3 (492 bp) (SEQ ID NO:8); Rex-1 gene,forward 5′-AGCAAGACGAGGCAAGGCCAGTCCAGAATA-3′ (SEQ ID NO:9), reverse5′-GAGGACACTCCAGCATCGATAAGACACCAC-3′ (423 bp) (SEQ ID NO: 10) andα-tubulin gene, forward 5′-CACCCGTCTTCAGGGCTTCTTGGTTT-3′ (SEQ ID NO:11), reverse 5′-CATTTCACCATCTGGTTGGCTGGCTC-3′(527bp) (SEQ ID NO: 12).PCR products were separated on 1.5% agarose gel and visualized byethidium bromide staining.

1. An in vitro method of producing a culture of non-human mammalianembryonic stem (ES) cells, comprising: (a) obtaining a blastomere from anon-human mammalian embryo; (b) culturing said blastomere with ES orembryonic carcinoma (EC) cells to form ES cell colonies containing EScell originating from said blastomere; and (c) isolating and culturingthe ES cells originating from said blastomere.
 2. The method of claim 1,wherein the blastomere is obtained from an embryo prior to compaction ofthe morula.
 3. The method of claim 1, wherein the blastomere is obtainedfrom an embryo during compaction of the morula.
 4. The method of claim1, wherein the blastomere is obtained by partially or completelyremoving the zona pellucida surrounding the embryo.
 5. The method ofclaim 1, wherein the blastomere is cultured with embryo carcinoma cells.6. The method of claim 1, wherein the blastomere is cultured with EScells.
 7. The method of claim 1, wherein the blastomere is cultured withfactors that inhibit differentiation of ES cells.
 8. The method of claim1, wherein recombinant Oct-4 is introduced into the blastomere.
 9. Themethod of claim 1, wherein the ES cells of step (c) express an ES cellmarker protein.
 10. The method of claim 1, wherein the blastomereundergoes cell division and one progeny cell is used for genetic testingand a different progeny cell is used to produce an ES cell.
 11. A methodof producing a non-human mammalian embryonic stem (ES) cells, comprisingthe steps of: (a) obtaining a blastomere from a non-human mammalianembryo; (b) aggregating the blastomere with ES cells; (c) culturing theaggregated blastomere and ES cells until ES cells derived from theblastomere form; and (d) isolating the ES cells derived from theblastomere.
 12. A method of producing an ES cell line comprising thestep of culturing the ES cells produced by the method of claim 1, toproduce an ES cell line.
 13. The method of claim 11, wherein theblastomere is obtained from an embryo prior to compaction of the morula.14. The method of claim 11, wherein the blastomere is obtained from anembryo during compaction of the morula.
 15. The method of claim 11,wherein the blastomere is obtained by partially or completely removingthe zona pellucida surrounding the embryo.
 16. The method of claim 11,wherein the blastomere is cultured with factors that inhibitdifferentiation of the ES cells.
 17. The method of claim 11, whereinrecombinant Oct-4 is introduced into the blastomere.
 18. The method ofclaim 11, wherein at least one ES cell derived from the blastomereexpresses an ES cell marker protein.
 19. The method of claim 11, whereinat least one ES cell derived from the blastomere is used for genetictesting.
 20. The method of claim 11, wherein the embryo is cryopreservedafter step (a).
 21. The method of claim 20, wherein the embryo iscultured before being cryopreserved.
 22. The method of claim 1, whereinthe embryo is cryopreserved after step (a).
 23. The method of claim 22,wherein the embryo is cultured before being cryopreserved.
 24. A methodof producing an ES cell line comprising the step of culturing the EScells produced by the method of claim 11 to produce an ES cell line. 25.A method of producing a culture of non-human mammalian embryonic stem(ES) cells, comprising the steps of: (a) isolating a blastomere from an8-cell stage embryo by biopsy through a hole drilled in the zonapellucida or by zona-denuding and disaggregating said embryo; (b)culturing said blastomere with ES cells to form clumps of cells thatcomprise ES cells originated from said blastomere; and (c) isolating anddissociating said clumps of cells, and further isolating and culturingthe ES cells that originated from said blastomere to produce a cellculture of ES cells that originated from said blastomere.